Project Summary The classical estrogen receptor, ER, is a ligand regulated transcription factor important for normal development, homeostasis and reproduction that also contributes to pathological processes such breast cancer. It is generally accepted that coactivators contribute to positive regulation of ER activity, while corepressors are important for negative regulation of ERs bound to antagonists such as 4-hydroxytamoxifen (4HT); the latter interactions are important for determining the relative antagonist activity of selective estrogen receptor modulators (SERMs) such as 4HT. However, there is little information on the mechanisms by which corepressors regulate ER function in the absence of pharmacologic agents, and our recent results challenge the widely accepted view that the corepressors SMRT and NCoR exert their effects only on ER bound to pharmacological antiestrogens. Indeed, recent evidence points to a gene-selective requirement of SMRT for estrogen-stimulated, ER-dependent gene expression, and indicates that a change in our thinking about `corepressor' regulation of ER is required. Thus, the overall goal of this proposal is to comprehensively evaluate the role of the corepressors SMRT and NCoR with respect to their regulation of ER transcriptional activity in the presence of agonist as well as antagonist ligands, and to assess their mechanisms of action in these contexts. Our planned studies are based on four key observations. First, antagonists are not required for corepressors to bind to ER, nor are they required for corepressor interaction with ER target genes. Second, SMRT overexpression can enhance estradiol (E2)-stimulated ER activity, while depletion of SMRT expression can inhibit E2-ER activity. Third, SMRT and NCoR can bind to molecules required for efficient activation of gene expression. Fourth, a splice variant of SMRT which lacks its first repression domain and is expressed in tissues and cell lines, functions as a coactivator to stimulate the activity of ER bound to E2 as well as 4HT. These findings lead to the hypothesis that SMRT and NCoR play important roles in modulating ER function in the absence of pharmacological antiestrogens, and that the biological activities of these corepressors therefore contribute significantly to the ability of estrogen as well as antiestrogens to regulate ER transcriptional activity in a gene-specific manner. This hypothesis will be tested in three specific aims. 1) Determine the biological roles of SMRT and NCoR in regulating the expression of ER target genes. 2) Characterize novel SMRT and NCoR functional interactions required to regulate ER transcriptional activity. 3) Elucidate the molecular mechanisms by which SMRT and NCoR and their associated proteins contribute to positive and negative regulation of ER target gene expression. These studies will utilize state-of-the-art technologies to regulate the expression of SMRT, the SMRT splice variant and NCoR in cell models of ER action, develop new reagents and cell lines for corepressor functional analyses, and determine the molecular mechanisms through which corepressors control ER action. Project Narrative Understanding and exploiting normal and pharmacological regulation of estrogen receptor activity is of major significance to human health with respect to both preventing and treating estrogen-sensitive pathologies such as osteoporosis and breast cancer. Our novel findings on the requirement of SMRT for maximal estrogen stimulation of ER transcriptional activity are not in line with current expectations regarding corepressor regulation of ER function, and this strongly suggests that a paradigm shift in our understanding of the functional relationships between the SMRT/NCoR and ER is required. It is anticipated that a greater understanding of the complex control of ER function by corepressors will provide novel insights into mechanisms that selectively regulate ER in normal (e.g. tissue selectivity) and pathophysiological scenarios (e.g. breast cancer), and ultimately provide new approaches for identification and characterization of ER-based therapeutics.